Affinity chromatography devices

ABSTRACT

The present invention is directed to affinity chromatography devices that separate a targeted protein or antibody from an aqueous mixture containing the targeted protein or antibody. The chromatography device may contain a stacked membrane assembly or a wound membrane assembly. The membrane assemblies include at least one polymer membrane that contains therein inorganic particles. The polymer membrane and/or the inorganic particles have an affinity ligand bonded thereto. The affinity ligand may be a protein, an antibody, or a polysaccharide that reversibly binds to the targeted protein or antibody. The chromatography device may be repeatedly used and may be cleaned with a caustic solution between uses. The chromatography devices may have a dynamic binding capacity (DBC) of at least 30 mg/ml (or 0.07 micromol/ml) at 10% breakthrough at a residence time of 20 seconds or less.

This application is a Divisional Application under 35 U.S.C. § 121 ofU.S. patent application Ser. No. 15/094,428, filed on Apr. 8, 2016,which claims priority to U.S. Provisional Patent Application No.62/194,620 filed on Jul. 20, 2015, the entire contents of which areincorporated by reference in their respective entireties.

FIELD

The present disclosure relates generally to affinity chromatography, andmore specifically to chromatography devices containing a multilayeredmembrane assembly that enables the separation of a targeted protein orantibody from an aqueous mixture.

BACKGROUND

Chromatographic methods generally are used to separate and/or purifymolecules of interest such as proteins, nucleic acids, andpolysacchandes from a mixture. Affinity chromatography specificallyinvolves passing the mixture over a matrix having a ligand specific(i.e. a specific binding partner) for the molecule of interest bound toit. Upon contacting the ligand, the molecule of interest is bound to thematrix and is therefore retained from the mixture. Affinitychromatography provides certain advantages over other types ofchromatography. For example, affinity chromatography provides apurification method that can isolate a target protein from a mixture ofthe target protein and other biomolecules in a single step in highyield.

Despite the advantages of current affinity chromatography devices, thereexists a need in the art for a chromatography device that can be used atshorter residence times than conventional devices while providing thesame binding capacity or better binding capacities than currentofferings and that is re-useable.

SUMMARY

One embodiment relates to an affinity chromatography device thatincludes a housing, a first flow distributor and a second flowdistributor positioned at opposing ends of the housing, an inlet topermit fluid flow into the housing, an outlet to permit fluid flow outof the housing, and a stacked membrane assembly disposed within thehousing. The stacked membrane assembly includes two or more polymermembranes that contain therein first inorganic particles having a firstnominal particle size and second inorganic particles having a secondnominal particle size. At least one of the polymer membranes, the firstinorganic particles, and the second inorganic particles has covalentlybonded thereto an affinity ligand that reversibly binds to a targetedprotein or antibody. In one or more embodiment, the first and secondinorganic particles are of the same particle type. The inorganicparticles have a nominal particle size that may be about 5 microns,about 10 microns, about 15 microns, about 20 microns, about 25 micronsand combinations thereof.

Another embodiment relates to an affinity chromatography device thatincludes a housing, a first flow distributor and a second flowdistributor positioned at opposing ends of the housing, an inlet topermit fluid flow into the housing, an outlet to permit fluid flow outof the housing, and a wound membrane assembly disposed within thehousing. The housing may be a cylindrical housing. The wound membraneassembly includes at least one polymer membrane that contains thereinfirst inorganic particles having a first nominal particle size andsecond inorganic particles having a second nominal particle size. Atleast one of the polymer membrane, the first inorganic particles, andthe second inorganic particles has covalently bonded thereto an affinityligand that reversibly binds to a targeted protein or antibody. In oneor more embodiment, the first and second inorganic particles are of thesame particle type. The inorganic particles have a nominal particle sizethat may be about 5 microns, about 10 microns, about 15 microns, about20 microns, about 25 microns and combinations thereof.

Yet another embodiment relates to an affinity chromatography device thatincludes a housing, a first flow distributor and a second flowdistributor positioned at opposing ends of the housing, an inlet topermit fluid flow into the housing, an outlet to permit fluid flow outof the housing, and a stacked membrane assembly disposed within thehousing. The stacked membrane assembly includes two or more polymermembranes that contain therein inorganic particles having a nominalparticle size. At least one of the polymer membranes and the inorganicparticles has covalently bonded thereto an affinity ligand thatreversibly binds to a targeted protein or antibody. In at least oneembodiment, the polymer membranes are of the same polymer type.

A further embodiment relates to an affinity chromatography device thatincludes a housing, a first flow distributor and a second flowdistributor positioned at opposing ends of the housing, an inlet topermit fluid flow into the housing, an outlet to permit fluid flow outof the housing, and a wound membrane assembly. The wound membraneassembly includes a single polymer membrane containing therein inorganicparticles having a single nominal particle size. At least one of thepolymer membrane and the inorganic particles has covalently bondedthereto an affinity ligand that reversibly binds to a targeted proteinor antibody. The inorganic particles have a nominal particle size thatmay be about 5 microns, about 10 microns, about 15 microns, about 20microns, about 25 microns and combinations thereof. In one embodiment,the polymer membrane is a polytetrafluoroethylene membrane.

Another embodiment relates to an affinity chromatography device thatincludes a housing, a first flow distributor and a second flowdistributor positioned at opposing ends of the housing, an inlet topermit fluid flow into the housing, an outlet to permit fluid flow outof the housing, and a stacked membrane assembly disposed within thehousing. The stacked membrane assembly includes a first polymer membranecontaining therein first inorganic particles having a first nominalparticle size and a second polymer membrane containing therein secondinorganic particles having a second nominal particle size. At least oneof the first polymer membrane, the second polymer membrane, and theinorganic particles has covalently bonded thereto an affinity ligandthat reversibly binds to a targeted protein or antibody.

A further embodiment relates to a method for separating a target proteinor antibody from an aqueous mixture that includes passing an aqueousmixture through a chromatography device that includes a housing, a firstflow distributor and a second flow distributor positioned at opposingends of the housing, an inlet to permit fluid flow into the housing, anoutlet to permit fluid flow out of the housing, and a stacked membraneassembly disposed within the housing. The membrane assembly includes twoor more polymer membranes that contain therein first inorganic particleshaving a first nominal particle size second inorganic particles having asecond nominal particle size. At least one of the polymer membranes, thefirst inorganic particles, and the second inorganic particles hascovalently bonded thereto an affinity ligand that reversibly binds to atargeted protein or antibody. In one or more embodiment, the first andsecond inorganic particles are of the same particle type.

Yet another embodiment relates to a method for separating a targetprotein or antibody from an aqueous mixture that includes passing anaqueous mixture through a chromatography device that includes a housing,a first flow distributor and a second flow distributor positioned atopposing ends of the housing, an inlet to permit fluid flow into thehousing, an outlet to permit fluid flow out of the housing, and a woundmembrane assembly disposed within the housing. The wound membraneassembly includes at least one polymer membrane that contains thereinfirst inorganic particles having a first nominal particle size andsecond inorganic particles having a second nominal particle size. Atleast one of the polymer membrane, the first inorganic particles, andthe second inorganic particles has covalently bonded thereto an affinityligand that reversibly binds to a targeted protein or antibody. In oneor more embodiment, the first and second inorganic particles are of thesame particle type.

Another embodiment relates to a multi-well affinity chromatographydevice that includes a plurality of wells and a stacked membraneassembly disposed within at least one of the wells. The stacked membraneassembly includes two or more polymer membranes that contain thereinfirst inorganic particles having a first nominal particle size andsecond inorganic particles having a second nominal particle size. Atleast one of the polymer membranes, the first inorganic particles, andthe second inorganic particles has covalently bonded thereto an affinityligand that reversibly binds to a targeted protein or antibody. In oneor more embodiment, the first and second inorganic particles are of thesame particle type.

Yet another embodiment relates to a multi-well affinity chromatographydevice that includes a plurality of wells and a wound membrane assemblydisposed within at least one of the wells. The wound membrane assemblyincludes at least one polymer membrane that contains therein firstinorganic particles having a first nominal particle size and secondinorganic particles having a second nominal particle size. At least oneof the polymer membrane, the first inorganic particles, and the secondinorganic particles has covalently bonded thereto an affinity ligandthat reversibly binds to a targeted protein or antibody. In one or moreembodiment, the first and second inorganic particles are of the sameparticle type.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the disclosure and are incorporated in and constitute apart of this specification, illustrate embodiments, and together withthe description serve to explain the principles of the disclosure.

FIG. 1 is an exploded view of a stacked membrane assembly containingpolymer membranes having therein inorganic particles according to atleast one embodiment;

FIG. 2 is a schematic illustration of layers of polymer membranes withina membrane assembly in accordance with an exemplary embodiment;

FIG. 3 is an exploded view of polymer membranes having an alternatingconfiguration within a stacked membrane assembly according to at leastone exemplary embodiment;

FIG. 4 is an exploded view of a chromatography device containing astacked membrane assembly in accordance with an exemplary embodiment;

FIG. 5 is a schematic illustration of a cross-section of achromatography device containing a stacked membrane assembly accordingto an exemplary embodiment;

FIG. 6 is a schematic illustration of a cross-section of achromatography device containing a wound membrane assembly having apolymer membrane in accordance with an embodiment;

FIG. 7 is a schematic illustration of a cross-section of achromatography device containing a wound membrane assembly having twopolymer membranes in an alternating configuration in accordance with anembodiment;

FIG. 8 is an exploded view of a chromatography device containing aspirally wound membrane assembly in accordance with exemplaryembodiments;

FIG. 9A is a schematic illustration of a multi-well plate in accordancewith an embodiment of the invention;

FIG. 9B is a schematic illustration of a portion of the multi-well platedepicted in FIG. 9A showing a portion of a stacked membrane assemblypositioned on a porous substrate according to at least one exemplaryembodiment;

FIG. 10 is a graphical illustration of the dynamic binding capacity(DBC) at twenty (20) second residence time at 10% breakthrough(milligrams IgG bound per milliliter bed volume) of variouschromatography devices; and

FIG. 11 is a graphical illustration of the dynamic binding capacity(DBC) at sixty (60) second residence time at 10% breakthrough(milligrams IgG bound per milliliter bed volume) of two comparativechromatography devices.

DETAILED DESCRIPTION

Persons skilled in the art will readily appreciate that various aspectsof the present disclosure can be realized by any number of methods andapparatus configured to perform the intended functions. It should alsobe noted that the accompanying figures referred to herein are notnecessarily drawn to scale, but may be exaggerated to illustrate variousaspects of the present disclosure, and in that regard, the figuresshould not be construed as limiting. It is to be understood that, asused herein, the term “on” is meant to denote an element, such as apolymer membrane, is directly on another element or intervening elementsmay also be present.

The present invention is directed to affinity chromatography devicesthat separate a targeted protein or antibody from an aqueous mixturecontaining the targeted protein or antibody. The chromatography devicecontains a membrane assembly that includes at least one polymermembrane, such as a fluoropolymer membrane, that contains thereininorganic particles. An affinity ligand may be bonded to the inorganicparticles and/or to the polymer membrane. The chromatography device maybe repeatedly used and may be cleaned with a caustic solution betweenuses. In addition, the chromatography devices have a dynamic bindingcapacity (DBC) of at least 30 mg/ml at 10% breakthrough at a residencetime of 20 seconds or less in devices where an Fc binding protein is theaffinity ligand. In chromatography devices where an antibody, a non-Fcbinding protein, or a polysaccharide is the affinity ligand, thechromatography devices have a dynamic binding capacity (DBC) of at least0.07 micromol/ml at 10% breakthrough.

The membrane assemblies described herein include at least one polymermembrane that contains therein inorganic particles. The polymermembranes may contain from about 20 mass % to about 95 mass %, fromabout 35 mass % to about 90 mass %, or from about 50 mass % to about 85mass % inorganic particles. Non-limiting examples of suitable inorganicparticles include silica, zeolites, hydroxyapatite, metal oxides, andcombinations thereof. The inorganic particles may have a nominalparticle size of about 0.1 microns, about 0.5 microns, about 1 micron,about 5 microns, about 10 microns, about 15 microns, about 20 microns,or about 25 microns or more. Additionally, the inorganic particles maybe either solid or porous and may have a variety of sizes and shapes.Further, the inorganic particles may be monodisperse or polydisperse.

In an exemplary embodiment, the affinity ligand is covalently bonded tothe inorganic particles. In another embodiment, the affinity ligand iscovalently bonded to the polymer membrane. In a further embodiment, theaffinity ligand may be bound to both the polymer membrane and theinorganic particle(s). The affinity ligand may be a protein, antibody,or polysaccharide that reversibly binds to a targeted protein orantibody. In one embodiment, the affinity ligand is a protein thatreversibly binds, for example, to an Fc region of an antibody, anantibody fragment, an Fc fusion protein, or an antibody/drug conjugate.In another embodiment, the affinity ligand is an antibody, Protein L, ora polysaccharide that reversibly binds to a protein or a proteinfragment to which it is specific. Exemplary affinity ligands for use inthe affinity chromatography device include, but are not limited to,Protein A, Protein G, Protein L, human Fc receptor protein, antibodiesthat specifically bind to other proteins, and heparin. The affinityligand may be native, recombinant, or synthetic. In yet anotherembodiment, the affinity ligand is a metal affinity ligand thatreversibly binds to His-Tagged Proteins.

In one embodiment, the membrane assembly includes at least one polymermembrane that contains therein inorganic particles where the polymermembranes are positioned in a stacked or layered configuration to form astacked membrane assembly. The term “stacked membrane assembly” is meantto denote a chromatographic article that contains at least two polymermembranes positioned such that one polymer membrane is on anotherpolymer membrane. The polymer membranes may be positioned in a stackedconfiguration by simply laying the membranes on top of each other. FIG.1 depicts one exemplary orientation of a stacked membrane assembly 10that includes polymer membranes 20 containing therein inorganicparticles having a nominal particle size. It is to be appreciated thatthe inorganic particles are described herein with respect to nominalparticle size to take into consideration the variability of sizes andshapes of the inorganic particles. The arrow 5 depicts the direction offluid flow through the membrane assembly 10.

In one exemplary embodiment, the polymer membrane 20 contains a singletype of inorganic particle having a single nominal particle size. Forinstance, the polymer membrane 20 may contain therein porous silicaparticles that have a nominal particle size of about 20 microns. It isto be understood that the term “silica” as used herein is meant todenote silicon dioxide that does not contain any measurable amount ofboron or contains no boron as measured by x-ray photoelectronspectroscopy (XPS).

Alternatively, the polymer membrane 20 may contain more than one type ofinorganic particle and/or more than one nominal particle size within thepolymer membrane 20. In other words, the polymer membrane 20 may containat least first inorganic particles and second inorganic particles wherethe first inorganic particles are different from the second inorganicparticles in nominal particle size and/or type. For example, the polymermembrane 20 may include a mixture of a first particle size (e.g., 20microns) and a second particle size (e.g., 10 microns) of the same ordifferent inorganic particle (e.g., porous silica). The mixture ofinorganic particles within the polymer membrane 20 may be any mixture,such as a 50/50 blend, a 30/70 blend, a 60/40 blend, a 25/75, or a 20/80blend.

The polymer membranes 20 in the stacked membrane assembly 10 may bepositioned such the first and second polymer membranes are separatedfrom each other by a distance (d), as shown schematically in FIG. 2. Thedistance d may range from about 0 microns to about 50 microns, fromabout 0 microns to about 25 microns, from about 0 microns to about 10microns, or from about 0 microns to about 5 microns. In someembodiments, the distance d is zero or substantially zero microns (i.e.,less than or equal to 0.1 microns). The distance may also be less thanabout 50 microns, less than about 25 microns, less than about 10microns, less than about 5 microns, less than about 1 micron, or zeromicrons.

In another embodiment depicted generally in FIG. 3, the stacked membraneassembly 10 includes a first polymer membrane 20 containing inorganicparticles having a first nominal particle size and a second polymermembrane 30 containing inorganic particles having a second nominalparticle size. The first polymer membrane 20 and the second polymermembrane 30 may be stacked in an alternating fashion to form themembrane assembly 10, such as is exemplified in FIG. 3. The first andsecond polymer membranes 20, 30 may alternatively be stacked in anon-alternating configuration. For instance, multiple first membranes 20may be positioned on multiple second membranes 30. In anotherembodiment, multiple polymer membranes 20 may be alternatively stackedwith multiple second membranes 30 to form the membrane assembly 10.Also, a plurality of first polymer membranes 20 may be alternativelylayered on a single (or lesser or greater number of) second polymermembrane 30, and vice versa, to form a stacked membrane assembly 10.Additional polymer membranes containing inorganic particles may also bepresent in the membrane assembly.

The polymer membranes 20, 30 may be positioned in a stackedconfiguration by simply laying the membranes on top of each other.Alternatively, the polymer membranes 20, 30 may be stacked andsubsequently laminated together with heat and/or pressure or otherconventional methods. Embodiments employing two polymer membranes thatare co-expanded to produce a composite membrane assembly is alsoconsidered to be within the purview of the invention. Such a compositemembrane assembly may contain two (or more) layers of polymer membranesthat may be co-extruded or integrated together. In exemplaryembodiments, the first polymer membrane 20 and second polymer membrane30 are in a stacked configuration and the distance between the first andsecond polymer membranes is zero or substantially zero.

In another embodiment, the inorganic particles are of the same type inboth the first polymer membrane 20 and the second polymer membrane 30.For example, both polymer membranes 20, 30 may include porous silicaparticles. Alternatively, the inorganic particles in the first andsecond membranes may have different nominal particle sizes. In someembodiments, the inorganic particles in the first polymer membrane 20and the second polymer membrane 30 have the same nominal particle sizeor substantially the same nominal particle size.

In a further embodiment, the first and/or second polymer membranes 20,30 may contain more than one type of inorganic particle within thepolymer membrane. In other words, the first polymer membrane 20 and/orthe second polymer membrane 30 may contain at least a first inorganicparticle and a second inorganic particle where the first inorganicparticle is different from the second inorganic particle in nominalparticle size and/or type. For example, the polymer membrane(s) 20, 30may include a 50/50 mixture of a first nominal particle size (e.g., 20microns) and a second nominal particle size (e.g., 10 microns) of thesame or different inorganic particle. The mixture of inorganic particleswithin the first and/or second polymer membrane 20, 30 may be anymixture, such as, for example, a 50/50 blend, a 30/70 blend, a 60/40blend, a 25/75, or a 20/80 blend. In at least one embodiment, themembrane assembly 10 includes first and second polymer membranes 20, 30that are formed of the same polymer membrane (e.g., PTFE membranes) andthe inorganic particles are the same (e.g., porous silica particles) butthe inorganic particles have different nominal particle sizes (e.g., 20micron silica particles in one polymer membrane and 10 micron silicaparticles in the other polymer membrane).

The polymer membranes 20, 30 discussed herein may be formed of the sameor different polymer(s). In one or more exemplary embodiment, at leastone of the polymer membranes is a fluoropolymer membrane. It is to beappreciated that one, or more than one, fluoropolymer membrane may formor form part of the stacked membrane assembly 10. The fluoropolymermembranes may be derived from the same fluoropolymer source, fromdifferent sources, or a combination thereof. In at least one exemplaryembodiment, the fluoropolymer membrane is a polytetrafluoroethylene(PTFE) membrane or an expanded polytetrafluoroethylene (ePTFE) membrane.Expanded polytetrafluoroethylene (ePTFE) membranes prepared inaccordance with the methods described in U.S. Pat. No. 7,306,729 toBacino et al., U.S. Pat. No. 3,953,566 to Gore, U.S. Pat. No. 5,476,589to Bacino, or U.S. Pat. No. 5,183,545 to Branca et al. may be usedherein. Further, the fluoropolymer membrane may be rendered hydrophilic(e.g., water-wettable) using known methods in the art, such as, but notlimited to, the method disclosed in U.S. Pat. No. 4,113,912 to Okita, etal. A coating that effectively binds to a ligand, such as described inU.S. Pat. No. 5,897,955 to Drumheller, U.S. Pat. No. 5,914,182 toDrumheller, or U.S. Pat. No. 8,591,932 to Drumheller may be applied tothe polymer membrane.

The fluoropolymer membrane may also include an polymer materialcomprising a functional tetrafluoroethylene (TFE) copolymer materialwhere the functional TFE copolymer material includes a functionalcopolymer of TFE and PSVE (perfluorosulfonyl vinyl ether), or TFE withanother suitable functional monomer, such as, but not limited to,vinylidene fluoride (VDF), vinyl acetate, or vinyl alcohol. A functionalTFE copolymer material may be prepared, for example, according to themethods described in U.S. Pat. No. 9,139,707 to Xu et al. or U.S. Pat.No. 8,658,707 to Xu et al.

It is to be understood that throughout the application, the term “PTFE”is utilized herein for convenience and is meant to include not onlypolytetrafluoroethylene, but also expanded PTFE, expanded modified PTFE,and expanded copolymers of PTFE, such as described in U.S. Pat. No.5,708,044 to Branca, U.S. Pat. No. 6,541,589 to Baillie, U.S. Pat. No.7,531,611 to Sabol et al., U.S. Pat. No. 8,637,144 to Ford, and U.S.Pat. No. 9,139,669 to Xu, et al.

In one or more exemplary embodiment, the polymer membrane may be formedwith one or more non-fluoropolymer materials, such as, but not limitedto ultra-high molecular weight polyethylene as taught in U.S. PatentPublication No. 2014/0212612 to Sbriglia, polyparaxylylene as taught inU.S. Provisional Application No. 62/030,419 to Sbriglia, VDF-co-(TFE orTrFE) polymers as taught in U.S. Provisional Patent Application No.62/030,442 to Sbriglia, altemating poly(ethylene tetrafluoroethylenepolymers as taught in U.S. Provisional Patent Application No. 62/030,448to Sbriglia. Also, the polymer membrane may be, for example, apolyolefin membrane (e.g. polypropylene membrane), an organic membrane(e.g., a cellulose-based membrane), a structured hydrogel membrane, oran agarose membrane.

The total number of polymer membranes present in the stacked membraneassembly 10 is not particularly limited, and depends on the desired enduse and/or desired mass transit flow within the membrane assembly. Thestacked membrane assembly may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) total polymer membranes.It is to be appreciated that hundreds or even thousands of polymermembranes may be present in the stacked membrane assembly 10.

In addition, the polymer membranes present in the stacked membraneassembly 10 may have a thickness from about 1 micron to about 10,000microns, from about 100 microns to about 5,000 microns, from about 500microns to about 3,000 microns, or from about 650 microns to about 1,000microns. As used herein, the term “thickness” is the direction of thepolymer membrane normal to the length area of the polymer membrane.

The chromatography device 100 may further include a porous frit 40positioned at the top and/or bottom of the membrane assembly 10. Theporous frit 40, housing 50, and flow distributors 60, 70 may be formedof a thermoplastic polymer such as polypropylene, polyethylene, or otherpolyolefins. Alternatively, the porous frit 40 may be formed of aninorganic or metallic material, so long as the frit 40 does not hinderthe operation of the chromatography device.

Turning to FIGS. 4 and 5, the stacked membrane assembly 10 may bedisposed within a housing 50 having a first flow distributor 60 and asecond flow distributor 70 disposed at opposite ends of the housing 50.In exemplary embodiments, the housing 50 is cylindrical. In oneembodiment, the flow distributor 60 contains an impingement surface sothat the flow of the aqueous mixture is redirected 90 degrees from thefeed direction. This redirection prevents the flow from directlyimpinging on the polymer membrane and promotes a more uniform flowfront. The polymer membranes in the stacked membrane assembly 10 may beadhered to the housing 50 at the inner walls of the housing via anyconventional process (e.g., melt sealing or use of a sealant) thatprevents flow between the membrane periphery and the housing. The flowdistributors 60, 70 may be sealed to the housing 50 by a similar oridentical process. Each flow distributor 60, 70 includes an inlet 80 andan outlet 85, respectively, to permit the flow of an aqueous mixturethrough the affinity chromatography device 100. Specifically, the inlet80 permits fluid flow into the housing 50 and the outlet 85 permitsfluid flow out of the housing 50. In use, the aqueous mixture flowssequentially through the polymer membranes in the stacked membraneassembly 10 in the direction illustrated by arrow 5. As the aqueousmixture is passed through the chromatography device 100, the affinityligand reversibly binds to the targeted protein or antibody, therebyeffectively removing it from the aqueous mixture. The targeted proteinor antibody may be removed from the affinity ligand, for example, bypassing a fluid that has a lower pH through the device.

In a further embodiment, at least one polymer membrane having inorganicparticles therein (such as the polymer membrane(s) described above) iswrapped around a perforated hollow or solid core 150 to form a woundmembrane assembly 110. In one exemplary embodiment depicted generally inFIG. 6, the membrane assembly 110 contains a polymer membrane 20 thatincludes first inorganic particles having a first nominal particle sizeand second inorganic particles having a second nominal particle size.The first and second inorganic particles may be of the same type (e.g.,porous silica) or may be of different types (e.g., silica and zeolite),and may have the same or different nominal particle size(s). The mixtureof inorganic particles within the polymer membrane 20 may be anymixture, such as, for example, a 50/50 blend, a 30/70 blend, a 60/40blend, a 25/75, or a 20/80 blend.

In some embodiments, an intermediate film is positioned on the polymermembrane and is wrapped with the polymer membrane such that uponwinding, the intermediate film is situated between the wound layers ofthe polymer membrane. The intermediate film may be a fluoropolymer filmor a non-fluoropolymer film (e.g., a polypropylene or other polyolefinfilm). Additionally, the intermediate film may be porous or non-porous.

In another embodiment, the membrane assembly 110 may be formed of asingle polymer membrane 20 containing therein inorganic particles havinga single nominal particle size. For example, a single polymer membranethat is at least partially filled with inorganic particles with anominal particle size and having an affinity ligand bonded thereto maybe used as a membrane assembly 110. Additionally, multiple polymermembranes having therein inorganic particles of the same particle sizemay be used to form the membrane assembly 110.

Two (or more) polymer membranes may be present in the wound membraneassembly 110. When more than one polymer membrane is present in thewound membrane assembly 110, a first polymer membrane 20 having a firstnominal particle size and a second polymer membrane 30 having a secondnominal polymer size may be layered on each other in a stackedconfiguration and then wound about the core 150 in the stackedconfiguration to form the wound membrane assembly 110, such as isdepicted in FIG. 7. Alternatively, a first polymer membrane 20 may bewound around the core 150 and then a second polymer membrane 30 may besubsequently wrapped around the wound first polymer membrane 110. It isto be appreciated that if greater than two membranes is desired, thepolymer membranes 20, 30 may be stacked in an alternating ornon-alternating configuration as discussed above prior to winding.

As depicted in FIGS. 6 and 7, the wound membrane assembly 110 isdisposed within a housing 50 having a first flow distributor 60 and asecond flow distributor 70 disposed at opposite ends of the housing 50The chromatography device 200 includes an inlet 80 and an outlet 85 topermit the flow of an aqueous mixture through the affinitychromatography device 100. Specifically, the inlet 80 permits fluid flowinto the housing 50 and the outlet 85 permits fluid flow out of thehousing 50. In exemplary embodiments, the housing is cylindrical. Thewound membrane assembly 110 may further include at least one porous frit40 positioned normal to the wound membrane assembly 110 and adjacent toflow distributor 60 and/or flow distributor 70. In one embodiment, thecore 150 is solid and the flow distributor 60 contains an impingementsurface so that the flow of the aqueous mixture is redirected radiallyfrom the feed direction. In use, the aqueous mixture flows through thepolymer membrane(s) in the membrane assembly 110 in the directionillustrated by arrow 5, orthogonal to the areal thickness direction ofthe membrane. As the aqueous mixture is passed through thechromatography device 200, the affinity ligand reversibly binds to thetargeted protein or antibody, thereby effectively removing it from theaqueous mixture. The targeted protein or antibody may be removed fromthe affinity ligand, for example, by passing a fluid that has a lower pHthrough the device.

In an alternate embodiment, the core 150 is a hollow, porous core thatenables the aqueous mixture to flow outwardly from the core 150 andthrough the polymer membrane(s). Alternatively, the aqueous mixtureflows through the polymer membrane(s) inwardly and into the core 150.

In yet another embodiment, a polymer membrane, a multilayer stackedmembrane assembly, or a spiral wound membrane assembly as describedabove may be affixed to a multi-well plate 130 containing a poroussurface 90 separating a lower chamber that can be operated at reducedpressure from an upper chamber that is operated at a higher (e.g.,atmospheric) pressure. In the embodiment depicted in FIGS. 9A and 9B,the membrane assembly contains a plurality of polymer membranes 20having therein first inorganic particles having a first nominal particlesize and second inorganic particles having a second nominal particlesize. In operation, the aqueous mixture flows normal to the polymermembranes 20.

The affinity chromatography devices described herein have a dynamicbinding capacity (DBC) of at least 30 mg/ml at 10% breakthrough at aresidence time of 20 seconds or less where an Fc binding protein is theaffinity ligand. Where an antibody, a non-Fc binding protein, or apolysaccharide is the affinity ligand, the chromatography devices have adynamic binding capacity (DBC) of at least 0.07 micromol/ml at 10%breakthrough at a residence time of 20 seconds or less. In addition, thechromatography devices may be used multiple times without losingsubstantial dynamic binding capacity. Specifically, the chromatographydevices may be cleaned with a caustic solution (e.g. sodium hydroxide)after each separation process and reused.

Although exemplary embodiments of the membrane assemblies 10, 110 aredescribed herein, it is to be appreciated that any number of polymermembranes as well as any and all combinations of types of polymermembranes, types of inorganic particles, sizes of inorganic particles,shapes of inorganic particles, and orientations of the polymer membraneswithin the membrane assembly are within the scope of this disclosure.Also, some or all of the polymer membranes may vary in composition,thickness, permeability, etc. from each other.

Persons skilled in the art will readily appreciate that various aspectsof the present disclosure can be realized by any number of methods andapparatus configured to perform the intended functions. It should alsobe noted that the accompanying figures referred to herein are notnecessarily drawn to scale, but may be exaggerated to illustrate variousaspects of the present disclosure, and in that regard, the figuresshould not be construed as limiting.

Test Methods

It should be understood that although certain methods and equipment aredescribed below, other methods or equipment determined suitable by oneof ordinary skill in the art may be alternatively utilized.

Method for Determining the Dynamic Binding Capacity at 10% Breakthrough

The chromatography device was inserted in a AKTApurifier™ (GEHealthcare) liquid chromatography system's flow path and a single cycleconsisting of the following protocol was performed multiple times togenerate the data shown in FIG. 10. For the purpose of examining causticsolutions and their effect on dynamic binding capacity, only a causticclean in place (CIP) solution was used. Table 1 sets forth the solutionsutilized. Table 2 sets forth the protocol steps to determine the dynamicbinding capacity at 10% breakthrough.

TABLE 1 Solution Description A 50 mM Phosphate supplemented with 150 mMSodium Chloride, pH ~7.4 B 100 mM Citrate, pH ~3.5 CIP 0.1M NaOH Feed1.2-1.3 mg/mL polyclonal IgG (Lee Biosciences) dissolved in Solution AStorage 20/80 v/v ethanol/water

TABLE 2 Bed Volume/ Volumetric Flow Volume of Solution Used Rate =Seconds Step Solution (Number of Bed Volumes) Residence Time 1 A 6 20 2B 6 20 3 A 6 20 4 Feed Until Absorbance at 280 nm = 20 10% Breathrough 5A 3 20 6 B 10  20 7 A 3 20 8 CIP 15 minutes contact time at 1 N/A mL/min9 A 6 20 10 Water 6 20 11 Storage 6 20 12 Return to Step 1 and continuein this manner until the desired number of cycles is executed.

The dynamic binding capacity was determined for the above-describedchromatography devices in this manner. The dynamic binding capacity ofthe commercial devices were determined by the same method, but with thefollowing exception: The residence time for the commercial devices was60 seconds instead of 20 seconds.

Thus, the chromatography devices described herein were evaluated atthreefold faster volumetric flow rates than the commercial devices wereevaluated, with the exception of the CIP steps, which were identical.

EXAMPLES Example 1—Stacked Membranes

A porous polytetrafluoroethylene (PTFE) membrane having 15 mass percentPTFE and 85 mass percent porous silica particles (Grace, Baltimore, Md.)having a nominal particle size of 10 micron (Grace, Baltimore, Md.) wasobtained. Additionally, a porous PTFE membrane having 15 mass percentPTFE and 85 mass percent porous silica particles (Grace, Baltimore, Md.)having a nominal particle size of 20 micron was obtained. The poroussilica particles in the PTFE membranes were substantially the same withrespect to other chemical and physical characteristics such as chemicalcomposition, particle shape, nominal particle porosity, nominal particlepore dimensions, and nominal particle BET surface areas.

Table 3 lists some of the physical characteristics of the two porousPTFE membranes.

TABLE 3 Porous Nominal Mass Silica Nominal Porous Percent Nominal PorousPorous Silica Porous Mass Particle Membrane Membrane Gurley Pore PorousSilica Percent Size Thickness Density Number Size Membrane ParticlesPTFE (micron) (micron) (grams/cc) (sec) (nm) A 85 15 10 650 0.41 30 100B 85 15 20 650 0.42 15 100

Porous membranes A and B were used to manufacture affinitychromatography devices. A polypropylene flow distributor was affixed toone end of a polypropylene cylinder housing. A porous polypropylene fritwas placed in the housing. The desired number of PTFE membrane layerswere stacked on the polypropylene frit within the housing. (See Table4). A second porous polypropylene frit was placed on top of the PTFEmembrane stack. A second polypropylene flow distributor was affixed tothe end of the cylindrical housing opposite the first polypropylene flowdistributor. The chromatography device was sealed via a heating process.

TABLE 4 Membrane Thickness Orientation with Bed Intermediate DevicePorous Respect to Fluid Vol- Device Desig- Membrane Flow DirectionDuring ume Permeability, nation Used Characterization (mL) k × 10⁻¹² cm²C 10 layers of Same 3.4 133 membrane A D 5 layers of Same 3.5 182membrane A and 5 layers of membrane B E 5 layers of Same 3.5 195membrane A and 5 layers of membrane B F 5 layers of Same 3.5 201membrane A and 5 layers of membrane B G 10 layers of Same 3.5 301membrane B

The intermediate devices were then treated in the same manner as thedevice of Example 3 and as a result, Protein A was covalently bonded tothe stacked PTFE membranes (e.g., membrane assembly).

The affinity chromatography devices whose manufacture was describedabove were tested to evaluate their twenty (20) second residence timedynamic binding capacities using multiple cycles and using the protocoldescribed in the Test Methods set forth herein. The performance of eachof these affinity chromatography devices is shown in FIG. 10.

Two different commercial packed particle bed affinity chromatographydevices were obtained and tested in the same manner, with the exceptionthat they were evaluated at a sixty (60) seconds residence time. One ofthe commercial devices was packed with silica particles containingresidual boron (Commercial Device 1) and the other commercial device waspacked with agarose (Commercial Device 2). The performance of the twocommercial affinity chromatography devices is depicted in FIG. 11. Theaffinity chromatography devices were evaluated at threefold faster ratesthan the commercial affinity chromatography devices.

Example 2—Spiral Wound Membrane

Porous PTFE Membrane B from Example 1 was used to construct a spiralwound affinity chromatographic device. A length of PTFE Membrane B waswound about a solid core with a lathe and membrane tensioning memberuntil the diameter of the resulting wound membrane assembly was slightlygreater than the inner diameter of a cylindrical polypropylene housing.The wound membrane assembly was then cut to the desired length dimensionwith a cutting tool while the wound device assembly was rotating on thelathe. The wound membrane assembly was inserted within a properlydimensioned cylindrical polypropylene housing after the housing had beensplit down its length to enable insertion of the wound membraneassembly. Porous polypropylene frits and polypropylene distributors wereassembled at the opposing ends of the cylindrical housing. The devicewas sealed via a heating process.

Three sealed intermediate chromatography devices of 3.5 mL bed volumewere manufactured in this manner. These devices were constructed usingthe same polypropylene distributors and polypropylene frits that wereused in Example 1. The intermediate devices were then treated in thesame manner as the device of Example 3 and, as a result, Protein A wascovalently bonded to the wound membrane assembly.

It was discovered that when a test solution (water) flowed through thesechromatography devices at various volumetric flow rates, thepermeability of the wound membrane assembly was substantially greaterthan the permeability of the stacked membrane assembly of Example 1. Inthe devices containing the wound membrane assembly, the test solutionflowed orthogonal to the areal thickness direction of the membrane.

Example 3

This example illustrates one method for covalently binding Protein A toa porous PTFE membrane or multiple porous PTFE membranes that includePTFE and porous silica particles, where the porous membrane or multipleporous membranes was (were) integrated into a device housing having aninlet and an outlet for flowing fluids. Although this method isdescribed with respect to an affinity chromatography device thatcontains a stacked membrane assembly, it is to be appreciated that thismethod is applicable regardless of the orientation or configuration ofthe porous membrane relative to the fluid flow path and regardless ofthe particle shape, nominal particle size, nominal particle pore size ornominal particle pore volume of the porous silica particle phase, andwhether the membrane assembly is stacked or wound or otherwiseassembled.

All solutions were 0.2 micron filtered unless stated otherwise. Inaddition, all solutions were flowed through the devices with the aid ofa syringe pump or a peristaltic pump.

A 3.5 mL bed volume chromatography device was manufactured from a porouspolytetrafluoroethylene (PTFE) membrane having 15 mass % PTFE and 85mass % porous silica particles (Davisil® Silica Unbonded Grades,XWP1000A, 16-24 μm, Grace, Baltimore, Md.). A stacked membrane assemblywas produced. The membrane assembly was washed with 21 mL of a solutionof 95 parts by volume ethanol (Sigma-Aldrich, St. Louis, Mo.) and 5parts by volume deionized water (Neu-Ion, Inc., Baltimore, Md.) at avolumetric flow rate of 0.7 mL/min. Next, 10.5 mL of a non-filteredsolution of 5.885 grams of 3-glycidoxypropyltrimethoxy silane (G6720,UCT Specialties, LLC, Bartram, Pa.) were dissolved in 94.5 mL of asolution of 95 parts by volume ethanol and 5 parts by volume deionizedwater and flowed through the membrane assembly at a volumetric flow rateof 0.7 mL/min. The device was left standing for about seventeen hours atroom temperature. Then the device was heated to 90° C. and held at thattemperature for two hours, followed by cooling the device to roomtemperature for one hour, after which, the membrane assembly was washedwith 21 mL of a solution of 95 parts by volume ethanol and 5 parts byvolume deionized water at a volumetric flow rate of 0.7 mL/min.

The membrane assembly was then treated with a solution of sulfuric acidand deionized water, pH=0.8, by flowing 21 mL of the solution throughthe device assembly at a volumetric flow rate of 0.7 mL/min, followed byheating at 90 degrees centigrade for two hours and then cooling thedevice to room temperature for one hour, followed by washing the treatedmembrane with 42 mL of 10 mM acetate buffer, pH=4.2. The 10 mM acetatebuffer, pH=4.2 was prepared by combining 3,952 mL of deionized waterwith 40 mL of 1M acetic acid (Sigma-Aldrich, Saint Louis, Mo.) and 8 mLof 1M sodium hydroxide (Sigma-Aldrich, Saint Louis, Mo.). Then, 120 mLof 10 mM acetate buffer was combined with 6.417 grams of sodiumperiodate (Sigma-Aldrich, Saint Louis, Mo.) and 10.5 mL of this solutionwas flowed through the membrane assembly at 0.7 mL/min. The device wasthen left to react for ninety minutes at room temperature, followed byflowing through the membrane assembly, 21 mL of 10 mM acetate buffer,pH-=4.2. This was followed by flowing through the membrane assembly, 21mL of a 0.01M sodium carbonate buffer, pH=10.9 at a volumetric flow rateof 0.7 mL/min. The 0.01M sodium carbonate buffer, pH=10.9, was preparedby combining 1000 mL of deionized water with 1.06 grams of sodiumcarbonate (Sigma-Aldrich, Saint Louis, Mo.) and 5.84 grams of sodiumchloride (EMD Chemicals, Inc., Gibbstown, N.J.).

Next, 21 mL of a 4 mg/mL solution of Protein A was flowed through thedevice in a recirculating flow pattern at a volumetric flow rate of 0.7mL/min for about 17 hours at room temperature. 4 mg/mL solution ofProtein A was prepared by combining 202.4 mL of the pH=10.9 sodiumcarbonate buffer prepared earlier and 17.6 mL of a 50 mg/mL Protein Asolution (Repligen rSPA, Waltham, Mass.). After about 17 hours, theProtein A solution recirculation process was stopped and therecirculated solution's absorbance at 280 nm was measured and comparedto that of the freshly prepared 4 mg/mL Protein A solution.

The membrane assembly was then washed at a volumetric flow rate of 0.7mL/min with 21 mL of a second 0.01M sodium carbonate buffer, pH=10.5,which had been prepared by combining 1000 mL of deionized water, 1.06grams sodium carbonate and 58.4 grams sodium chloride. This was followedby another wash step using 21 mL of 0.01M sodium carbonate buffer,pH=10.9 at a volumetric flow rate of 0.7 mL/min. Then, 31.5 mL of a 1mg/mL sodium borohydride (Sigma-Aldrich, Saint Louis, Mo.) solution in0.01M sodium carbonate buffer was flowed through the membrane assemblyat a volumetric flow rate of 0.26 mL/min. This was followed by flowingthrough the membrane assembly 31.5 mL of a 0.05 M phosphate buffersolution, pH=7.4, at a volumetric flow rate of 0.7 mL/min. The 0.05 Mphosphate buffer solution, pH=7.4, had been prepared earlier bycombining 1000 mL of deionized water with 1.035 grams of sodiumphosphate monobasic monohydrate (Sigma-Aldrich, Saint Louis, Mo.),11.393 grams of sodium phosphate dibasic heptahydrate (Sigma-Aldrich,Saint Louis, Mo.) and 8.766 grams of sodium chloride. The membraneassembly was then washed with 21 mL of deionized water at a volumetricflow rate of 0.7 mL/min. Next, the membrane assembly was washed with 21mL of a solution of 20 parts by volume ethanol and 80 parts by volumedeionized water. The device was then equipped with inlet and outlet capsand stored at 4° C. to 8° C.

Example 4

This example illustrates a second method for covalently binding ProteinA to a porous PTFE membrane or multiple porous PTFE membranes thatinclude PTFE and porous silica particles, where the porous membrane ormultiple porous membranes was (were) integrated into a device housinghaving an inlet and an outlet for flowing fluids. Although this methodis described with respect to an affinity chromatography device thatcontains a stacked membrane assembly, it is to be appreciated that thismethod is applicable regardless of the orientation or configuration ofthe porous membrane relative to the fluid flow path and regardless ofthe particle shape, nominal particle size, nominal particle pore size ornominal particle pore volume of the porous silica particle phase, andwhether the membrane assembly is stacked or wound or otherwiseassembled.

This method is different from the method described in Example 3 in thefollowing aspects. An aldehyde silane (PSX1050, UCT Specialties, LLC,Bartram, Pa.) was used instead of the epoxy silane of Example 3 A numberof other manufacturing steps were eliminated, as will be apparent tothose skilled in the art, upon comparison of this method and the methodof Example 3.

All solutions were 0.2 micron filtered unless stated otherwise. Allsolutions were flowed through the devices with the aid of a syringe pumpor a peristaltic pump.

A 3.5 mL bed volume chromatography device was manufactured from a porousmembrane sheet including polytetrafluoroethylene (PTFE) (15 masspercent) and porous silica particles (85 mass percent) having a nominalparticle size of 20 microns (Davisil® Silica Unbonded Grades, XWP1000A,16-24 μm, Grace, Baltimore, Md.). A stacked membrane assembly wasproduced. The membrane assembly was washed with 21 mL of a solution of95 parts by volume ethanol (Sigma-Aldrich, St. Louis, Mo.) and 5 partsby volume deionized water (Neu-Ion, Inc., Baltimore, Md.) at avolumetric flow rate of 0.7 mL/min. 10.5 mL of an unfiltered solution of3.21 grams of aldehyde silane (PSX1050, UCT Specialties, LLC, Bartram,Pa.) dissolved in 97.0 mL of a solution of 95 parts by volume ethanoland 5 parts by volume deionized water was flowed through the membraneassembly at a volumetric flow rate of 0.7 mL/min. The device was leftstanding for about seventeen hours at room temperature. Then the devicewas heated to 90° C. and held at that temperature for two hours,followed by cooling the device to room temperature for one hour, afterwhich, the membrane assembly was washed with 42 mL of a solution of 95parts by volume ethanol and 5 parts by volume deionized water at avolumetric flow rate of 0.7 mL/min. The membrane assembly was thenwashed with 21 mL of a 0.01M sodium carbonate buffer, pH=10.9 at avolumetric flow rate of 0.7 mL/min. The 0.01M sodium carbonate buffer,pH=10.9, was prepared by combining 1000 mL of deionized water with 1.06grams of sodium carbonate (Sigma-Aldrich, Saint Louis, Mo.) and 5.84grams of sodium chloride (EMD Chemicals, Inc., Gibbstown, N.J.).

Next, 21 mL of a 4 mg/mL solution of Protein A was flowed through thedevice in a recirculating flow pattern at a volumetric flow rate of 0.7mL/min for about 17 hours at room temperature. The 4 mg/mL solution ofProtein A was prepared by combining 202.4 mL of the pH=10.9 sodiumcarbonate buffer prepared earlier and 17.6 mL of a 50 mg/mL Protein Asolution (Repligen rSPA, Waltham, Mass.). After about 17 hours, theProtein A solution recirculation process was stopped and therecirculated solution's absorbance at 280 nm was measured and comparedto that of the freshly prepared 4 mg/mL Protein A solution.

The membrane assembly was then washed at a volumetric flow rate of 0.7mL/min with 21 mL of a second 0.01M sodium carbonate buffer, pH=10.5,which had been prepared by combining 1000 mL of deionized water, 1.06grams sodium carbonate and 58.4 grams sodium chloride. This washing wasfollowed by a wash step using 21 mL of 0.01M sodium carbonate buffer,pH=10.9 at a volumetric flow rate of 0.7 mL/min. Then 31.5 mL of a 1mg/mL sodium borohydride (Sigma-Aldrich, Saint Louis, Mo.) solution in0.01M sodium carbonate buffer was flowed through the device membraneassembly at a volumetric flow rate of 0.26 mL/min. This was followed byflowing through the device membrane assembly 31.5 mL of a 0.05 Mphosphate buffer solution, pH=7.4, at a volumetric flow rate of 0.7mL/min. The 0.05 M phosphate buffer solution, pH=7.4, had been preparedearlier by combining 1000 mL of deionized water with 1.035 grams ofsodium phosphate monobasic monohydrate (Sigma-Aldrich, Saint Louis,Mo.), 11.393 grams of sodium phosphate dibasic heptahydrate(Sigma-Aldrich, Saint Louis, Mo.) and 8.766 grams of sodium chloride.Then the device membrane assembly was washed with 21 mL of deionizedwater at a volumetric flow rate of 0.7 mL/min. Then the device membraneassembly was washed with 21 mL of a solution of 20 parts by volumeethanol and 80 parts by volume deionized water. The device was thenequipped with inlet and outlet caps and stored at 4° C. to 8° C.

The device prepared as described in this example was evaluated withpolyclonal IgG for one cycle and then it was evaluated with a crudeChinese Hamster Ovary (CHO) cell clarified culture medium (Aragen,Morgan Hills, Calif.) including a monoclonal antibody and impuritiessuch as host cell proteins. It was demonstrated that the device of thisexample was useful for purifying monoclonal antibodies from a crude CHOcell clarified culture medium.

Example 5—Stacked Membrane Device Including Porous Silica Mixture

A porous polytetrafluoroethylene (PTFE) membrane having 85 mass percentporous silica particles (Davisil® Silica Unbonded Grades, XWP1000A,16-24 μm, Grace, Baltimore, Md.) and 15 mass percent PTFE was obtained.The porous silica particles were present as a 50/50 by mass mixture oftwo different nominal particle sizes and these corresponded to theporous silicas used to produce porous membranes A and B in Example 1.

Table 5 lists some of the physical characteristics of the membraneobtained.

TABLE 5 Porous Mass silica Nominal Nominal percent nominal porous Porousporous porous Mass particle membrane membrane Gurley silica Poroussilica percent sizes thickness density Number pore size membraneparticles PTFE (micron) (micron) (grams/cc) (sec) (nm) C 85 15 10 & 20650 0.42 10 100

Porous PTFE membrane C was used to manufacture an affinitychromatography device. A polypropylene flow distributor was affixed toone end of a polypropylene cylinder housing. A porous polypropylene fritwas placed in the housing. The desired number of PTFE membrane layerswere stacked on the polypropylene frit within the housing. (See Table6). A second porous polypropylene frit was placed on top of the PTFEmembrane stack. A second polypropylene flow distributor was affixed tothe end of the cylindrical housing opposite the first polypropylene flowdistributor. The chromatography device was sealed via a heating process.

TABLE 6 Membrane thickness orientation with Bed Intermediate DevicePorous respect to fluid Vol- device Desig- membrane flow directionduring ume permeability, nation used characterization (mL) k × 10⁻¹² cm²10499664 12 layers of Same 3.6 303 membrane C

The intermediate device was then treated in the same manner as thedevice of Example 3 and as a result, Protein A was covalently bonded tothe membrane assembly.

The affinity chromatography device was then tested to evaluate its 10%dynamic binding capacity at twenty (20) second residence time using theprotocol described in Test Methods set forth herein. It was determinedthat the affinity chromatography device had a 10% dynamic bindingcapacity of 44 mg IgG per mL bed volume at 20 seconds residence time.

Example 6—Spiral Wound Membrane Device Including Porous Silica Mixture

Porous PTFE Membrane C from Example 5 was used to construct a spiralwound affinity chromatographic device. A length of PTFE Membrane C waswound about a solid core with a lathe and membrane tensioning memberuntil the diameter of the wound membrane assembly was slightly greaterthan the inner diameter of a polypropylene housing. The wound membraneassembly was then cut to the desired length with a cutting tool whilethe wound device assembly was rotating on the lathe. The desireddimensioned wound membrane assembly was inserted within a properlydimensioned cylindrical polypropylene housing after the housing had beensplit down its length to enable insertion of the wound membraneassembly. Porous polypropylene frits and polypropylene distributors wereassembled at the opposing ends of the cylindrical housing. The devicewas sealed via a heating process thereby producing an intermediatechromatography device of 3.5 mL bed volume.

The intermediate device was then treated in the same manner as thedevice of Example 3 and, as a result, Protein A was covalently bonded tothe wound membrane assembly. It was discovered that when a test solution(water) flowed through the affinity chromatography device at variousvolumetric flow rates, the permeability of the wound membrane assemblywas about twice the permeability of the stacked membrane assembly ofExample 5. In the device containing the wound membrane assembly, thetest solution flowed orthogonal to the areal thickness direction of themembrane. The affinity chromatography device had a 10% dynamic bindingcapacity of 31 mg IgG per mL bed volume at 20 seconds residence time.

The invention of this application has been described above bothgenerically and with regard to specific embodiments. It will be apparentto those skilled in the art that various modifications and variationscan be made in the embodiments without departing from the scope of thedisclosure. Thus, it is intended that the embodiments cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

What is claimed is:
 1. An affinity chromatography device comprising: ahousing; an inlet configured to permit fluid flow into the housing;first and second flow distributors, the first flow distributor and thesecond flow distributor positioned at opposing ends of the housing; anoutlet configured to configured to permit fluid flow out of the housing;and a stacked membrane assembly disposed within the housing, the stackedmembrane assembly comprising two or more polytetrafluoroethylenemembranes containing therein first inorganic particles having a firstnominal particle size and second inorganic particles having a secondnominal particle size, wherein at least one of: the first inorganicparticles, the second inorganic particles, or any combination thereofhas covalently bonded thereto affinity ligands comprising Fc bindingproteins, that reversibly bind to a targeted protein or antibody, andwherein the affinity chromatography device is configured to provide: adynamic binding capacity of at least 30 mg/ml at 10% breakthrough; and aresidence time of 20 seconds or less.
 2. The affinity chromatographydevice of claim 1, wherein the first and second inorganic particles arechosen from silica particles, zeolite particles, hydroxyapatiteparticles, metal oxide particles or any combination thereof.
 3. Theaffinity chromatography device of claim 1, wherein the two or morepolytetrafluoroethylene membranes are expanded polytetrafluoroethylenemembranes.
 4. The affinity chromatography device of claim 1, wherein theaffinity ligands are chosen from Protein A, Protein G, Protein L, humanFc receptor protein, antibodies, polysaccharides, or any combinationthereof.
 5. The affinity chromatography device of claim 1, wherein thefirst nominal particle size and the second nominal particle size arechosen from 0.1 microns, 0.5 microns, 1 micron, 5 microns, 10 microns,15 microns, 20 microns, 25 microns, or any combination thereof.
 6. Theaffinity chromatography device of claim 1, wherein the first nominalparticle size is different from the second nominal particle size.
 7. Theaffinity chromatography device of claim 1, wherein the first and secondinorganic particles are of the same particle type.
 8. An affinitychromatography device comprising: a housing; an inlet configured topermit fluid flow into the housing; first and second flow distributors,the first flow distributor and the second flow distributor positioned atopposing ends of the housing; an outlet configured to permit fluid flowout of the housing, and a stacked membrane assembly disposed within thehousing, the stacked membrane assembly comprising two or morepolytetrafluoroethylene membranes containing therein inorganic particlesof the same type having a nominal particle size, wherein the inorganicparticles have covalently bonded thereto affinity ligands comprising Fcbinding proteins, that reversibly bind to a targeted protein orantibody, and wherein the affinity chromatography device is configuredto provide: a dynamic binding capacity of at least 30 mg/ml at 10%breakthrough and; a residence time of 20 seconds or less.
 9. Theaffinity chromatography device of claim 8, wherein the nominal particlesize is chosen from 0.1 microns, 0.5 microns, 1 micron, 5 microns, 10microns, 15 microns, 20 microns, or 25 microns.
 10. The affinitychromatography device of claim 8, wherein the two or morepolytetrafluoroethylene membranes are expanded polytetrafluoroethylenemembranes.
 11. The affinity chromatography device of claim 8, whereinthe inorganic particles are chosen from silica particles, zeoliteparticles, hydroxyapatite particles, metal oxide particles, or anycombination thereof.
 12. The affinity chromatography device of claim 8,wherein the affinity ligands are chosen from Protein A, Protein G,Protein L, human Fc receptor protein, antibodies that specifically bindto other proteins, polysaccharides, or any combination thereof.
 13. Theaffinity chromatography device of claim 8, wherein the inorganicparticles are first inorganic particles having a first nominal particlesize, and wherein each of the at least two polytetrafluoroethylenemembranes further comprise second inorganic particles having a secondnominal particle size.
 14. The affinity chromatography device of claim8, wherein the second inorganic particles have covalently bonded theretoaffinity ligands that reversibly bind to a targeted protein or antibody.15. The affinity chromatography device of claim 8, wherein the first andsecond inorganic particles are chosen from: silica particles, zeoliteparticles, hydroxyapatite particles, metal oxide particles, or anycombination thereof.
 16. The affinity chromatography device of claim 8,wherein the affinity ligands are chosen from: Protein A, Protein G,Protein L, human Fc receptor protein, antibodies, polysaccharides, orany combination thereof.
 17. The affinity chromatography device of claim13, wherein the first nominal particle size and the second nominalparticle size are chosen from: 0.1 microns, 0.5 microns, 1 micron, 5microns, 10 microns, 15 microns, 20 microns, 25 microns, or anycombination thereof.
 18. The affinity chromatography device of claim 13,wherein the first nominal particle size is different from the secondnominal particle size.
 19. The affinity chromatography device of claim8, wherein the first and second inorganic particles are of the sameparticle type.